Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography

Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXF...

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Vydáno v:	IUCrJ Ročník 8; číslo 6; s. 878 - 895
Hlavní autoři:	Pandey, Suraj, Calvey, George, Katz, Andrea M., Malla, Tek Narsingh, Koua, Faisal H. M., Martin-Garcia, Jose M., Poudyal, Ishwor, Yang, Jay-How, Vakili, Mohammad, Yefanov, Oleksandr, Zielinski, Kara A., Bajt, Sasa, Awel, Salah, Doerner, Katarina, Frank, Matthias, Gelisio, Luca, Jernigan, Rebecca, Kirkwood, Henry, Kloos, Marco, Koliyadu, Jayanath, Mariani, Valerio, Miller, Mitchell D., Mills, Grant, Nelson, Garrett, Olmos, Jose L., Sadri, Alireza, Sato, Tokushi, Tolstikova, Alexandra, Xu, Weijun, Ourmazd, Abbas, Spence, John C. H., Schwander, Peter, Barty, Anton, Chapman, Henry N., Fromme, Petra, Mancuso, Adrian P., Phillips, George N., Bean, Richard, Pollack, Lois, Schmidt, Marius
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	England International Union of Crystallography 01.11.2021 International Union of Crystallography (IUCr)
Témata:	antibiotic resistance Binding ceftriaxone Crystal structure Crystallography Diffusion coefficient Diffusion rate drug discovery enzyme kinetics enzyme mechanisms Enzymes european x-ray free-electron laser irreversible inhibition Ligands megahertz pulse-repetition rate mix-and-inject serial crystallography protein structure determination Repetition Research Papers serial femtosecond crystallography substrate diffusion in crystals Substrates sulbactam Time lag Tuberculosis X ray sources x-ray crystallography β-lactamases β-lactamases megahertz pulse-repetition rate irreversible inhibition enzyme kinetics enzyme mechanisms sulbactam X-ray crystallography ceftriaxone mix-and-inject serial crystallography drug discovery antibiotic resistance substrate diffusion in crystals European X-ray Free-Electron Laser protein structure determination serial femtosecond crystallography
ISSN:	2052-2525, 2052-2525
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Popis
Shrnutí:	Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme–ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.
Bibliografie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 AC52-07NA27344 USDOE
ISSN:	2052-2525 2052-2525
DOI:	10.1107/S2052252521008125